OVERALL DESCRIPTION (adapted from the application): This is an application about further pursuing the use of inhibitors of cysteine proteases for the arrest of replication and attenuation of in vivo infectivity of the intracellular parasites Trypanosoma cruzi and Leishmania. In the previous TDRU funding period, these investigators showed that treatment with vinyl sulfone-derivatized peptidomimetics can rescue mice from a lethal T. cruzi infection, and in some cases eliminate any detectable parasites, achieving parasitological cure. A series of additional synthetic inhibitors, through collaborations with several members of the present multiproject application, were evaluated and found to be effective in the nM and low microM range against the T. cruzi cysteine protease cruzain. Several studies were performed to analyze the metabolites of the inhibitors generated in vivo, and a T. cruzi strain resistant to protease inhibitors was produced in culture. In addition, the mechanism of T. cruzi growth arrest by cysteine protease inhibitors was investigated, and concluded to be related to the accumulation of unprocessed cruzain in the parasite's Golgi apparatus and ER. The present application will be focused on screening additional compounds for enhanced efficacy and specificity against recombinant cruzain, and then examining activity of the most effective inhibitors on parasite replication and differentiation, using culture assays involving the three life cycle stages of T. cruzi. Ultrastructural studies to analyze the cellular effect of the inhibitors are also proposed. The best inhibitor leads identified in these previous steps will be subsequently evaluated in infectivity assays in mice. Essentially the same studies are proposed for Leishmania major, L. donovani and L. mexicana, but in this case the targeting will be on cathepsin B and L homologues identified in L. major. Since deletion mutants in some members of the cysteine protease family are available in L. mexicana, collaborative studies will be performed with this species to investigate the possible differential targeting of inhibitors to different gene family members.